Published online before print November 29, 2004, doi: 10.1161/01.CIR.0000148825.99184.50
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(Circulation. 2004;110:3544-3552.)
© 2004 American Heart Association, Inc.
Heart Failure |
Overexpression of Wild-Type Heat Shock Protein 27 and a Nonphosphorylatable Heat Shock Protein 27 Mutant Protects Against Ischemia/Reperfusion Injury in a Transgenic Mouse Model
From the Department of Medicine (J.M.H., D.D.B., B.T.S., E.S., W.H.D.), University of California, San Diego; the Cardiovascular Institute (J.L.M.), Loyola University Medical Center, Maywood, Ill; and the Department of Physiology (V.K.), Loyola University, Maywood, Ill.
Correspondence to Wolfgang H. Dillmann, MD, University of California, San Diego, Department of Medicine, 9500 Gilman Dr, La Jolla, CA 92093-0618. E-mail wdillman{at}ucsd.edu
Received December 5, 2003; de novo received July 20, 2004; revision received August 31, 2004; accepted September 8, 2004.
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Abstract |
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Background— The small heat shock protein 27 (hsp27) increases in expression with ischemia/reperfusion (I/R) insult in the heart. One feature of the small hsps is their ability to oligomerize and form intracellular aggregates. Oligomerization pattern is governed by the phosphorylation state of the protein that may influence their ability to protect against cellular stresses.
Methods and Results— We generated transgenic (tg) mice that overexpress a wild-type human hsp27 (hsp27tg) protein or a mutant hsp27 protein (mut-hsp27tg), in which serine residues (aa15, aa78, and aa82) were replaced by alanine residues, rendering them incapable of phosphorylation. Using a Langendorff perfusion model and an intraventricular balloon, we subjected hearts to 20 minutes of ischemia followed by 1 hour of reperfusion. During reperfusion, negative and positive pressure derivatives as well as developed pressures were significantly higher in both hsp27tg and mut-hsp27tg compared with control (P<0.01) mice, with no significant difference between hsp27tg and mut-hsp27tg. Creatine kinase release during reperfusion was higher in control compared with both hsp27tg and mut-hsp27tg (P<0.05). Malondialdehyde content as well as protein oxidation products were lower in mut-hsp27tg compared with control (P<0.05). hsp27tg hearts possessed oligomers that ranged in size from small to large, whereas mut-hsp27tg hearts contained no small oligomers.
Conclusions— These results indicate that in a tg mouse model, overexpression of either wild-type hsp27 or a nonphosphorylatable hsp27 mutant was equally capable of protecting the heart from I/R injury. Furthermore, the phosphorylation status of hsp27 may influence its ability to decrease oxidative stress.
Key Words: ischemia • reperfusion • free radicals
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Introduction |
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Myocardial ischemia/reperfusion (I/R) insult has been shown to induce cellular damage, such as membrane lipid peroxidation, disruption of cytoskeletal structure, disturbance of cellular oxidoreductive (redox) status, enzyme inactivation and misfolding, deterioration of mitochondrial function, and impairment of excitation-contraction coupling.1–6 Although it has become clear that heat shock proteins (hsps) have cardioprotective effects against myocardial I/R, the effect that hsp27 has in the intact heart has not been examined.7 The 27-kDa hsp, hsp27 (hsp25 in rodents), is part of a family of proteins that bind denatured proteins in times of stress to prevent aggregation.8 The human hsp27 gene encodes a 199–amino acid polypeptide whose expression can increase several-fold in response to a number of stimuli, including heat shock, simulated ischemia, and oxidative stress.9–12
Small hsps are characterized by their phosphorylative capacity, and they form multimeric structures. The human hsp27 protein contains 3 serine residues, located at amino acid positions 15, 78, and 82, which are phosphorylated in response to stress.13 The rodent form of the protein, hsp25, contains 2 phosphorylatable serine residues located at amino acid positions 15 and 86.14 Phosphorylation events are catalyzed by mitogen-activated protein kinase-activated protein kinase (MAPKAP) kinase 2/3 and p38-regulated/activated protein kinase (PRAK) kinase, both of which are regulated by the p38 MAPK pathway.12,15 Aggregate size becomes smaller as the level of hsp27 phosphorylation increases, with the phosphorylated form of the protein concentrated in small and medium-sized oligomers, and the nonphosphorylated form concentrated in larger oligomers.12,16,17 Induction of hsp27 by stressing agents or mitogens in stable Chinese hamster cell lines that constitutively express wild-type hsp27 caused a reduction in the multimeric size of the protein, which was not observed in an identical cell line expressing a nonphosphorylatable hsp27 mutant.18 Similarly, adult rat cardiomyocytes expressing a nonphosphorylatable hsp27 mutant formed larger oligomeric complexes than did wild-type, hsp27-infected cells.12 Phosphorylative capacity and the resulting aggregation patterns may influence the mode and cytoprotective ability afforded by hsp27 expression during cellular stress; however, conflicting results remain.19 Overexpression of wild-type hsp27 in rodent cells enhanced growth factor–induced F-actin accumulation after mitogenic stimulation, which is a positive effect linked to increased actin filament dynamics.20 In contrast, overexpression of a nonphosporylatable hsp27 mutant inhibited this response.20 These results were confirmed by observing a decreased thermotolerance in stable Chinese hamster cell lines expressing a nonphosphorylatable hsp27 mutant.18 In contrast, hsp25 nonphosphorylatable mutants conferred better protection than did wild-type hsp25 in L929 cells subjected to tumor necrosis factor (TNF)-
– and H2O2-induced cytotoxicity.19 Martin et al12 observed that overexpression of wild-type hsp27 or a nonphosphorylatable hsp27 mutant was equally capable of protecting adult rat myocytes from ischemic insult. These conflicting results may be due in part to the use of different cell culture types and the variability in hsp27 protein levels between studies.
hsp27 has been implicated in the decrease of oxidative stress resulting from cellular insult. Expression of hsp27 in L929 cells submitted to TNF- induction led to a decrease in reactive oxygen species (ROS) and an increase in cellular glutathione levels.21 Furthermore, hsp27-expressing cells possessed less lipid peroxidation and fewer protein oxidation products resulting from TNF- treatment, indicating an enhanced resistance to oxidative insult.21 Mehlen et al22 have suggested that the unphosphorylated form of hsp27, responsible for large oligomeric structures, is capable of protecting against oxidative stress by preserving glutathione levels. Similar results have been observed in L929 cells overexpressing a mutant hsp25 protein that is compromised in its phosphorylation ability, when compared with cells overexpressing wild-type hsp25.19
Much of the previous work has been performed in cell culture models, making it difficult to predict the effects of hsp27 overexpression in the intact heart presented with a global I/R insult. Furthermore, endogenous hsp25 levels are lower in adult cardiac myocytes in comparison with neonatal myocytes, indicating that increased hsp25 (or hsp27) may be advantageous to the adult heart presented with an ischemic episode.23 Thus, the goal of this study was to examine whether overexpression of either a wild-type human hsp27 protein or a nonphosphorylatable hsp27 mutant protein in a transgenic (tg) mouse model was capable of protecting the heart from stress associated with global I/R injury. To further our understanding of the effects of hsp27 phosphorylation, we attempted to determine whether overexpression of either protein afforded better protection from global I/R insult. Our results indicate that both proteins preserved contractile function and decreased creatine kinase (CK) release after global I/R in the heart with equal efficacy. In addition, the nonphosphorylated form of the protein was more effective at decreasing oxidative stress associated with I/R injury, indicating that a decrease in oxidative stress by-products may be involved in its protective mechanism of action.
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hsp27 and Mutant hsp27 (mut-hsp27) Transgenic Mouse Development
The animal experiments in this study conformed to the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and were approved by the University of California, San Diego, Animal Subjects Program. hsp27tg mouse lines were generated by using a chimeric transgene consisting of the human hsp27 gene inserted into the vector pCAGGS, as previously described.24,25 In brief, the pCAGGS vector places the hsp27 gene under the control of the human cytomegalovirus immediate-early enhancer and chicken ß-actin promoter with first intron.26 The 0.8-kb human hsp27 cDNA was inserted into the XhoI cloning site of pCAGGS as a SacI/HindIII fragment of
800 bp (Figure 1). The chimeric tg was cut out of the plasmid by SspI and BamHI digestion, purified, and used to generate the tg mice. The construct was given to the University of California, San Diego’s Transgenic Core facility, where the pronuclei of eggs from superovulated BALB/c mice crossed with C57/BL6 male mice were injected with 1 to 2 pL of the purified DNA fragment at a concentration of 2 µg/mL and transferred into the oviduct of pseudopregnant BALB/c mice. Litters were delivered after 19 to 20 days of gestation. mut-hsp27tg mouse lines were generated as stated by using similar techniques and cloning strategies. Serines 15, 78, and 82 were altered to alanines by site-directed mutagenesis, as previously described by Landry et al.10 The mut-hsp27tg construct was examined previously in an adenoviral expression system and shown to form large oligomeric complexes.12 Several lines of animals were generated for both the hsp27tg and mut-hsp27tg mouse lines, and lines with similar gene expression levels were used for the experimental procedures. To verify whether the chimeric tg was present in the genome, DNA from 3-week-old mice was isolated from tail clips and examined by Southern blotting. Southern blots were analyzed with a 32P-labeled 800-bp probe generated from the chicken ß-actin promoter with first intron. All control and tg mice were generated on a CB6F1 mouse background, and experimental procedures were performed on animals 5 months old.
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Northern Blot Analysis
Isolation of tissue RNA for Northern blotting was performed as described by Chomczynski and Sacchi.27 Ten micrograms of total RNA from each sample was subjected to Northern blot analysis, as previously described.28,29 Hybridization was performed with random-primed cDNA probes as per the manufacturer’s instructions (Amersham). Blots were then exposed to film, and autoradiographic signals were assessed.
hsp27 Protein Analysis
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 12% gel as described by Laemmli, with equal amounts of protein loaded for each study treatment.30 Relative amounts of hsp25 proteins were determined by using an anti-hsp25 rabbit antibody (product No. SPA-801, Stressgen). The secondary antibody was an anti-rabbit IgG horseradish peroxidase conjugate (product No. NA934V, Amersham). Relative amounts of combined hsp27 and hsp25 proteins were determined by using an anti-hsp25/27 antibody that reacts with both human (hsp27) and mouse (hsp25) proteins (product No. MAB3842, Chemicon). The secondary antibody was an anti-mouse IgG horseradish peroxidase conjugate (product No. NA931V, Amersham). Detection of signal was performed according to the NEN Renaissance ECL detection system manufacturer’s directions (NEN Life Sciences). Blots were then exposed to film, and autoradiographic signals were assessed.
hsp27 Native Analysis and Sucrose Gradient Electrophoresis
Native PAGE analysis was performed as previously described.12 Equal amounts of proteins (7 µg) were loaded on a 5% polyacrylamide native PAGE gel (Bio-Rad) and fractionated for 300 V-h. Immunoanalysis was performed as described in the previous paragraph. For sucrose gradients, frozen heart samples were homogenized in 10 mmol/L HEPES-buffered saline (pH 7.4) containing leupeptin, aprotinin, p-aminoethyl benzenesulfonyl fluoride hydrochloride, sodium orthovanadate, and 0.5% Triton X-100 before clarification at 16 000g. After quantification, 100 µg of protein was loaded on a 2-mL sucrose gradient (5% to 40%) and spun at 166 180gav for 5 hours with a TLS 55 rotor. Fractions (100 µL) were collected from the top and either precipitated with trichloroacetic acid before resuspension in 30 µL Laemmli buffer or mixed with Laemmli buffer directly before denaturation and analysis by 12% SDS-PAGE for immunoanalysis as described.
Langendorff-Perfused Heart Protocol
Hearts were isolated and transferred to a miniaturized Langendorff setup for contractile studies as previously described.31 In brief, hearts were removed from anesthetized mice and immersed in cold cardioplegic solution. After cannulation of the aorta, the hearts were perfused retrogradely at 37°C with a modified Krebs-Henseleit buffer (in mmol/L: 118 NaCl, 4.7 KCl, 2.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 0.5 Na2EDTA, and 5.5 glucose). A small fluid-filled balloon was inserted into the left ventricular cavity and coupled to a pressure transducer (Millar Instruments). The balloon was inflated until the end-diastolic pressure (EDP) reached 10 mm Hg. Platinum wires were placed on the surface of the right atrium and used to pace the hearts at 400 beats/min. Hearts were perfused for 15 minutes to achieve stable cardiac function and then placed through an ischemic period in which pacing and perfusion were ceased for 20 minutes. During this time, hearts remained submerged in a 37°C jacketed chamber to maintain temperature. After the no-flow ischemic period, pacing and perfusion were reinitiated and continued for 1 hour. Digitized recordings of ventricular pressure and its first derivative were captured on an IBM-compatible PC with the use of Windaq software. Left ventricular peak systolic pressure (PSP), EDP, maximum speed of contraction (+dP/dt), and maximum speed of relaxation (–dP/dt) were analyzed from the recordings.
CK Release
CK release was used as a determinant of cellular disruption and damage, as previously described.32 CK activities were measured spectrophotometrically at 340 nm with a commercially available system (Sigma) in which release was determined as the activity in the perfusate at various times during reperfusion (1, 2, 3, 4, 5, 10, 30, and 60 minutes). CK activities were expressed as micromoles of NADH formed per minute per gram. Protein content was assessed with the Bradford method, with bovine serum albumin as the standard.33
Lipid Peroxidation
Peroxidation of lipids was assessed by measurement of malondialdehyde (MDA), a stable end-product derived from the oxidation of polyunsaturated fatty acids and related esters, as previously described.32 MDA content was assayed spectrophotometrically at 586 nm with use of a lipid peroxidation assay kit (Calbiochem).
Protein Oxidation
Oxidatively modified proteins were examined by measuring carbonyl groups introduced into protein side chains. Carbonyl-containing proteins were cross-reacted with 2,4-dinitrophenylhydrazone (DNPH) to produce DNP adducts. These derivatized proteins were separated by Western blotting, as described earlier,30 and then probed for DNP moieties with a specific rabbit anti-DNP antibody (product No. S7150, Serologicals Corp). A goat anti-rabbit IgG (horseradish peroxidase conjugated) was used as a secondary antibody, and detection of signal was assessed as above. Experiments were performed several times to confirm accuracy of the results. To confirm oxidative modification of proteins, derivatized carbonyl groups were measured spectrophotometrically at 390 nm on all samples as previously described.34 Results are expressed as nanomoles of protein carbonyls per milligram protein.
Statistics
Means and standard errors were calculated for all data sets. Data were analyzed by 1-way ANOVA, and, when appropriate, Fisher’s least significant difference post hoc tests were performed (Systat version 5.03). P<0.05 was considered significant.
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Characterization of hsp27 and mut-hsp27tg Animals
hsp27tg and mut-hsp27tg mice were examined for tg expression in the heart. hsp27tg and mut-hsp27tg animals possessed large quantities of hsp27 mRNA, whereas control animals had none (Figure 2A). hsp27tg animals possessed slightly higher amounts of hsp27 mRNA compared with mut-hsp27tg animals, which may have been due in part to a difference in tg copy number. Analysis of hsp27 mRNA was performed with a cDNA probe coding for hsp25, which enabled detection of both endogenous hsp25 and exogenous hsp27 species. Examination of the mRNA results indicated that the relative abundance of the endogenous hsp25 species was many-fold less than that of the hsp27 tg (Figure 2A). No significant differences were observed in the levels of another small hsp,
B-crystallin, in either tg strain compared with control (data not shown). For protein analysis, 2 different antibodies were used to assess either hsp25 protein content or the combined hsp25 and hsp27 protein contents. Using an antibody that reacts with both hsp25 and hsp27, we determined the combined hsp25/hsp27 protein contents (Figure 2B top). Control hearts possessed modest amounts of hsp25 protein (lanes 1–3), in contrast to hsp27tg (lanes 4–6) and mut-hsp27tg (lanes 7–9) hearts, which contained very large amounts of combined hsp25 and hsp27 protein (Figure 2B top). Using an hsp25-specific antibody, we found that control hearts (lanes 1–3) contained a little less hsp25 protein compared with both hsp27tg hearts (lanes 4–6) and mut-hsp27tg (lanes 7–9) hearts (Figure 2B bottom). We used densitometric analysis and the Image J program (Image Jv.1.29x, NIH) to gain some insight into the fold differences in both hsp25 and hsp27 protein in the 3 groups. hsp27tg hearts possessed 100-fold greater hsp25/hsp27 protein content compared with control hearts, of which only a 0.3-fold increase was hsp25 protein. mut-hsp27tg hearts possessed 76-fold greater hsp25/hsp27 protein content compared with control hearts, of which only a 0.13-fold increase was hsp25 protein. Thus, very large amounts of hsp27 protein content could be observed in both tg lines, with minimal increases observed in hsp25 protein content. These protein results are similar to those observed for our mRNA analysis (Figure 2A). We examined heart weight–body weight ratios in adult animals to determine whether hsp27tg or mut-hsp27tg caused any hypertrophic abnormalities. No significant differences were noted between any of the groups (Table).
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Functional Recovery After I/R Injury in hsp27tg and mut-hsp27tg Animals
To evaluate whether overexpression of either hsp27tg or mut-hsp27tg was capable of preserving contractile function, we used a global no-flow I/R model with a Langendorff perfusion apparatus and inserted a balloon into the left ventricle. Baseline (aerobically perfused, non-I/R) functional values before initiation of the I/R protocol are summarized in the Table. No significant differences were observed in +dP/dt, –dP/dt, PSP, EDP, and total developed pressure (DP) in any group (Table). Hearts were subjected to 20 minutes of no-flow ischemia, after which flow was reinitiated and continued for 1 hour, resulting in 75% to 80% decreases in +dP/dt, –dP/dt, and DP in the control group (see the Table versus Figures 4 and 5
). Both +dP/dt (Figure 3A) and –dP/dt (Figure 3B) were significantly greater in both hsp27tg and mut-hsp27tg animals compared with control (P<0.01 for both). No differences were observed between hsp27tg and mut-hsp27tg hearts, indicating that both proteins were equally capable of preserving function relative to control hearts (Figure 3A and 3B). In addition, DP was significantly greater in both hsp27tg and mut-hsp27tg hearts after global, no-flow I/R compared with control (Figure 4C, white boxes, P<0.01). hsp27tg and mut-hsp27tg overexpression was equally capable of preserving pressure development after I/R compared with control (Figure 3C, white boxes). However, analysis of PSP revealed significantly increased values in mut-hsp27tg compared with control and hsp27tg animals after I/R (Figure 3C, axis, P<0.01). In contrast, hsp27tg animals had a significantly lower EDP after I/R compared with control and mut-hsp27tg animals (Figure 3C, black boxes, P<0.01). Thus, the increased DPs observed in both tg lines occurred through either an increase in PSP (mut-hsp27tg) or a decrease in EDP (hsp27tg).
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P<0.01, DP for hsp27tg and mut-hsp27tg vs control. 
P<0.01, PSP for mut-hsp27tg vs hsp27tg and control. All values are represented as mean±SEM, n=10 for each group. Abbreviations are as defined in text.
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Decreased Cellular Damage After I/R Injury in hsp27tg and mut-hsp27tg Animals
Cellular damage was examined in hsp27tg and mut-hsp27tg animals at various times after I/R insult by measuring CK release in cardiac effluents. Preischemic basal release of CK did not differ between groups. CK release was significantly greater in control animals after ischemia at 1, 2, 5, 10, and 30 minutes of reperfusion compared with hsp27tg and mut-hsp27tg animals (Figure 4, P<0.05 for all). No significant differences were observed in CK release after I/R between hsp27tg and mut-hsp27tg animals (Figure 4). These results indicate that overexpression of either hsp27tg or mut-hsp27tg was equally capable of protecting against I/R-initiated cellular disruption and damage in the myocardium.
Decreased Oxidative Stress After I/R Injury in mut-hsp27tg Animals
Oxidative stress has been implicated as a potential contributor to cellular damage during I/R injury.2,35,36 As a result, we examined indicators of oxidative stress after global I/R in the myocardium. Baseline (aerobically perfused, non-I/R) MDA values were between 1 and 2 µmol · L–1 · mg protein–1 for all 3 groups, with no significant differences existing between any group. After I/R, mut-hsp27tg animals had a significantly lower MDA content compared with hsp27tg and control animals (Figure 5, P<0.05). Carbonyl-containing DNP adducts were measured by Western immunoanalysis on several hearts from each treatment group. mut-hsp27tg animals possessed lower amounts of protein oxidation products compared with both control and hsp27tg hearts (Figure 6A). To further confirm the qualitative differences observed in the Western immunoanalysis, we measured the protein carbonyl-containing DNP adducts spectrophotometrically on all of the samples from the 3 treatment groups. Baseline (aerobically perfused, non-I/R) protein oxidation values were between 0.4 and 0.6 nmol/mg protein for all 3 groups, with no significant differences existing between any group. As observed in the Western blot analyses, mut-hsp27tg animals possessed a smaller amount of protein carbonyl products compared with both control and hsp27tg animals (Figure 6B). Taken together, these results indicate that the mut-hsp27tg animals were more capable of coping with the oxidative stress occurring during global I/R in the heart than both hsp27tg and control animals.
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hsp27tg and mut-hsp27tg Oligomerization Patterns
The phosphorylation status of hsp27 has been suggested as having a role in its structural organization, which manifests in the oligomerization patterns of the protein. As a result, we examined the oligomerization pattern of hsp27tg and mut-hsp27tg mice before and after global I/R in the heart by 2 different methods. Protein extracts from hsp27tg and mut-hsp27tg mouse hearts were subjected to nondenaturing PAGE and probed for hsp27 protein content. Control hearts possessed no hsp27 protein (Figure 7A, lane 1). Because the primary antibody used was specific for hsp27, no endogenous hsp27 protein was observed. hsp27tg mice possessed hsp27 oligomers that ranged in size from small to large before I/R (Figure 7A, lanes 2 and 3). In contrast, mut-hsp27tg mice contained hsp27 oligomers of medium and large sizes only (Figure 7A, lanes 4 and 5). To further characterize the aggregation pattern of hsp27tg and mut-hsp27tg overexpression, we analyzed the hearts from tg mice subjected to global I/R. Again, control hearts subjected to I/R showed no hsp27 protein (Figure 7A, lanes 6 and 8). I/R hsp27tg hearts possessed primarily smaller hsp27 oligomers (Figure 7A, lanes 2 and 3 versus 7). In contrast, mut-hsp27tg hearts displayed a larger oligomeric distribution that shifted upward with I/R (Figure 7A, lanes 4 and 5 versus 9), whereas hsp27tg hearts shifted downward with I/R (Figure 7A, lanes 2 and 3 versus 7). To confirm the results seen in Figure 7A, we fractionated heart homogenates from the various treatment groups by sucrose gradient ultracentrifugation and then visualized hsp27 oligomerization patterns. By this method, hsp27tg hearts displayed hsp27 oligomers ranging in size from small to large before I/R (Figure 7B, 1). In contrast, mut-hsp27tg hearts displayed hsp27 aggregate patterns composed primarily of medium-sized oligomers before I/R (Figure 7B, 3). After I/R, the hsp27tg heart oligomerization patterns shifted primarily to smaller oligomers (Figure 7B, 2), whereas mut-hsp27tg hearts displayed hsp27 oligomers that were shifted to a larger complex profile (Figure 7B, 4). These results are in agreement with those observed in our native gel analysis (Figure 7A) and are in agreement with others indicating that phosphorylation status can influence hsp27 oligomeric organization.12,19
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The ramifications of myocardial I/R injury are widespread, imparting damage to multiple loci within the cell and its constituents.4,5 Protection from I/R insult is crucial, and it has become clear that hsps can exert cardioprotective effects against I/R-initiated damage.7 hsp27 is of particular interest because its expression has been associated with decreased generation of cellular ROS and increased glutathione levels, an important player in the antioxidant system.16,21 Because ROS are a central component of I/R-induced damage, we generated tg mice overexpressing human hsp27 and examined the impact on global I/R insult. Furthermore, to test the contribution of the phosphorylation of hsp27, we produced tg mice that overexpressed a nonphosphorylatable hsp27 protein for comparison. Several lines of animals were generated for each tg, and experiments were performed on lines with comparable protein levels based on Western blot analyses. Northern blot and Western blot analyses revealed large amounts of either hsp27 or mut-hsp27 proteins in the hsp27tg and mut-hsp27tg animals, respectively (Figure 2A and 2B top). The increases in hsp27 protein were in excess of 75- to 100-fold, which is in contrast to increases in hsp25 protein, which were only increased 0.13- to 0.3-fold (Figure 2A and 2B). It is unclear why a very small increase in hsp25 was observed, but it may in part be due to expression of human hsp27, which is being perceived as a stress to the cell. Examination of heart weight–body weight ratios in adult animals indicated that no obvious cardiac abnormality was apparent (Table).
The central focus of the current study was to determine whether overexpression of either hsp27tg or mut-hsp27tg protein could preserve contractile function after global, no-flow I/R in the heart. In both tg mouse lines, both +dP/dt and –dP/dt were significantly greater than in controls, indicating that expression of either protein species was equally effective at preserving contractile function (Figure 3A and 3B). Examination of DPs after global I/R revealed trends similar to those observed with +dP/dt and –dP/dt (Figure 3C). Interestingly, mut-hsp27tg animals had greater PSPs after I/R compared with hsp27tg and control animals (Figure 3C, P<0.01), whereas hsp27tg animals had lower EDPs after I/R compared with mut-hsp27tg and control animals (Figure 3C, P<0.01). Thus, the resulting increases seen in DP in both the hsp27tg and mut-hsp27tg animals occurred through distinctly different mechanisms. The results are puzzling and may be due to a number of different factors. The lower EDP seen in the hsp27tg hearts after global I/R may be due to decreased ischemic contracture.37 Such a condition would favor a lessening in so-called rigor bridges and preserve cross-bridge cycling, indicating that the phosphorylated form of the protein was more capable of protecting structural proteins (actin, microfilaments) in the cell compared with the nonphosphorylated form of the protein, as had been suggested previously.19,38 The increased PSP seen in the mut-hsp27tg hearts is more difficult to explain and may be due to a general protection of contractile function due in part to decreased oxidative stress and preservation of glutathione levels in the cell.19,22
Oxidative stress by-products increase in response to myocardial I/R insult.39–43 As a result, we examined MDA and protein oxidation after global I/R. MDA formation was significantly lower in the mut-hsp27tg hearts compared with control and hsp27tg hearts after global I/R (Figure 5). MDA content analyses were performed with a specific chromogenic reagent that reacts with MDA and is less susceptible to interfering agents typical of classic thiobarbituric acid–reacting substances assays. Nevertheless, other methods of lipid peroxidation assessment exist, such as high-pressure liquid chromatography, mass spectrometric measurement of F2-isoprostanes, and stable aldehydes (4-hydroxynonenal), and it is possible that utilization of an alternative method might have yielded different results. Measurement of carbonyl adducts in proteins by Western blotting revealed qualitatively fewer carbonyl adducts in mut-hsp27tg hearts compared with both hsp27tg and control hearts (Figure 6A). Confirmation of these findings spectrophotometrically indicated that mut-hsp27tg hearts had significantly fewer protein carbonyls than did control hearts (Figure 6B). Because of the variability in the hsp27tg hearts, this finding appeared as a trend when compared with mut-hsp27tg animals (Figure 6B). It should be pointed out that carbonyl content constitutes only one oxidative protein modification and that other forms of protein modification exist, such as oxohistidine and dityrosine, and as such, assessment of these parameters might have yielded different results. Nevertheless, these results indicate that the nonphosphorylated form of hsp27, responsible for the larger aggregates, may exert its protective effects through decreasing oxidative stress in response to global I/R. Such a finding is in agreement with others who have found similar results in cell culture models, wherein the nonphosphorylated form of either hsp27 or hsp25 is expressed.19,22 Mehlen et al22 have suggested that a direct relation between the intracellular level of glutathione and the structural organization of hsp27 exists and that hsp27 "chaperone" activity plays an important role in mediating redox changes. Such a hypothesis would help to explain our observation that the nonphosphorylated form of hsp27 was more effective at reducing oxidative stress by-products.
Because the phosphorylation state of hsp27 influences its oligomerization pattern, we examined the aggregation pattern of hsp27tg and mut-hsp27tg tg mice before and after global I/R in the heart by native gel and sucrose gradient ultracentrifugation analyses (Figure 7). Before global I/R, hsp27tg hearts displayed multiple hsp27-specific bands of various sizes, whereas mut-hsp27tg hearts displayed bands of both medium and large sizes (Figure 7A). Our results are consistent with those of others who have examined overexpression of either hsp27 or hsp25 and their associated nonphosphorylatable mutants in cell culture models.12,19,22 Interestingly, hearts subjected to global I/R showed banding patterns similar to those before I/R, but these effects appeared to be somewhat accentuated, with hsp27tg hearts possessing a majority of smaller aggregates and mut-hsp27tg possessing larger aggregates (Figure 7A). These results were confirmed by analysis of hearts subjected to sucrose gradient ultracentrifugation. Before I/R, hsp27tg hearts showed hsp27 oligomerization patterns that ranged from small to large, in contrast to mut-hsp27tg hearts, which displayed hsp27 oligomers of mostly medium size (Figure 7B). After global I/R, both patterns were accentuated, with hsp27tg oligomers concentrated in smaller oligomers and mut-hsp27tg in larger oligomers (Figure 7B). These results suggest that the resistances to I/R observed in both the hsp27tg and mut-hsp27tg are potentially caused by different mechanisms. Our results are similar to those of Preville et al,19 who observed in L929 cells expressing a nonphosphorylatable hsp25 mutant a recovery of protein in a hyperaggregated form that was accentuated in response to TNF- and others who have shown that phosphorylation by stressing agents causes a reduction in the multimeric size of wild-type hsp27.18,44
In summary, we report for the first time that tg overexpression of hsp27 or a nonphosphorylatable hsp27 mutant protein was equally capable of protecting the mouse heart from global I/R insult but that the protection may be caused by different mechanisms or loci of action. Nonphosphorylatable hsp27 mutants decreased oxidative stress by-products with a greater efficacy compared with hsp27 tgs, indicating that attenuation of ROS or their effects may be the loci of action for this hsp27 isoform.
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Acknowledgments |
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This work was supported by NIH grant HL-49434 and the Midwest Affiliate of the American Heart Association (0235411Z to J.L.M.). Dr Hollander is a recipient of an NIH postdoctoral fellowship (5 T32 HL07444).
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Footnotes |
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The online-only Data Supplement, which contains an additional figure, can be found with this article at http://www.circulationaha.org.
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